Transient forebrain or global ischemia arising during cardiac arrest affects 200,000 Americans each year and in many cases results in delayed death of hippocampal neurons and severe cognitive deficits. To date, treatment of the neurodegeneration and cognitive deficits associated with global ischemia is an unmet need. REST (repressor element 1 silencing transcription factor) is a gene silencing transcription factor that is widely expressed during embryogenesis and plays a strategic role in the transition from pluripotent stem cells to neurons. During the late stages of neuronal differentiation, REST downregulation is critical to acquisition of the neural phenotype. A fundamental mechanism by which REST abundance is regulated in pluripotent cells and neural progenitors is via SCF (Skp1-Cul1-F-box protein)/?-TrCP-dependent, ubiquitin-based proteasomal degradation. We found that ischemia suppresses ?-TrCP in adult neurons, and that proteasomal inhibitors activate REST even in the absence of insult. Our targeted ChIP-on-chip analysis indicates that REST suppresses a discrete subset of target genes in postischemic neurons. The overall objectives are to understand how neuronal insults activate REST and examine a potential role for two newly identified REST targets, Kv7 and TRPV1, in the neurodegeneration associated with global ischemia. The central hypothesis driving the research is that global ischemia acts via ubiquitin proteasomal degradation to upregulate REST in adult neurons and that REST promotes silencing of a subset of transcriptionally-responsive target genes, which drives neuronal death. Specific Aims are: Aim 1. Identify the molecular mechanisms by which ischemia regulates REST in CA1 neurons by pharmacological and genetic approaches including a floxed REST knockout mouse. 1) Examine impact of ischemia on the ubiquitin E3 ligase ?-TrCP, proteasome activity and REST stability; 2) Examine ability of inhibitors of proteasomal degradation to increase REST abundance, enhance REST stability, silence REST targets and induce neuronal death in the absence of insult; 3) Examine ability of ?-TrCP knockdown and dominant-negative (dn)?-TrCP to enhance REST abundance and stability, silence REST targets and induce neuronal death; and 4) Examine ability of ?-TrCP and Cul1 overexpression to suppress REST upregulation and silencing of REST targets and promote survival of postischemic CA1 neurons. Aim 2. Determine whether novel REST targets identified by a targeted ChIP-on-chip analysis are altered functionally and causally related to neuronal death and identify mechanisms by which specificity of REST silencing is achieved. 1) Verify that candidate genes identified as positive hits exhibit epigenetic dysregulation (technical validation); 2) Document that epigenetic dysregulation results in alterations in gene expression, protein abundance and synaptic function (biological validation); 3) Document that novel REST targets Kv7 and TRPV1 are causally related to neuronal death by genetic manipulation; and 4) identify mechanisms by which specificity of REST silencing is achieved in insulted CA1 neurons. These translational studies will accelerate development of novel therapeutic strategies to ameliorate this serious cause of human morbidity and mortality.